Natural killer (NK) cells are a third subtype of lymphocytes besides B and T cells. NK cells provide an important immune function in the defense against viruses and other intracellular pathogens. Unlike B and T cells, NK cells do not exhibit specificity for antigen. They acquired their name because they can kill cells without prior stimulation by antigen. The killing of normal healthy cells is prevented by inhibitory receptors on NK cells that recognize major histocompatibility class I molecules. A large number of receptors that activate the cytotoxic response of NK cells have been described. However, it is not clear if any one of these receptors is sufficient and/or necessary to activate natural cytotoxicity. The major goal of this project is to define the molecular basis of NK cell activation, using normal, unmanipulated NK cells, and to characterize the receptors involved. A reconstitution system has been developed to test for the contribution of individual receptors to the NK cell activation. Several specific ligands of NK cell receptors were expressed in an insect cell line in order to define the requirements for activation of NK cell adhesion and cytotoxicity. Surprisingly, expression of ICAM-1 alone was sufficient to induce lysis of the insect cells, suggesting that the integrin LFA-1 is essential, not only for adhesion of NK cells to target cells, but also for transmitting activation signals. The ability of LFA-1 to provide an early signal for cytotoxicity was demonstrated by the activation of the guanine exchange factor Vav1 upon mixing of NK cells with insect cells expressing ICAM-1. Vav1 activation was independent of actin polymerization. As Vav1 is an important regulator of actin cytoskeleton remodelling during cytotoxicity, these data show that LFA-1 can signal upstream of actin polymerization. Expression of ligands for other NK cell activation receptors (such as CD16, CD2, and 2B4) was not sufficient to induce cytotoxicity, but their co-expression with ICAM-1 did enhance the lysis induced by ICAM-1 alone. The regulation of effector functions in resting NK cells is not well understood. The insect cell system was used to show that engagement of receptors CD2 and 2B4 by their respective ligand on target cells resulted in a strong enhancement of adhesion, even in the absence of cytokines or chemokines.